WO2025205612A1 - Method for propagating regulatory cells - Google Patents

Abstract

Disclosed are: a method for producing a cell population in which regulatory T cells have been propagated, the method comprising (1) a step for culturing a cell population containing primary regulatory T cells in the presence of interleukin (IL)-2, IL-4, IL-33, and a TNFR2 agonist; (1A) a method for propagating a cell population containing regulatory T cells, the method comprising a step for culturing a cell population containing primary regulatory T cells in the presence of IL-2, IL-4, IL-33, and a TNFR2 agonist; a cell population containing regulatory T cells obtained by the method; and a drug comprising the cell population containing regulatory T cells.

Description

Method for Proliferating Regulatory Cells

The present invention relates to a method for producing a cell population in which primary regulatory T cells (Treg) have expanded, a cell population containing regulatory T cells obtained by the method, a pharmaceutical product containing the cell population containing regulatory T cells, and the like.
BACKGROUND OF THE INVENTION

In recent years, research and development into cell therapy (Treg therapy) using regulatory T cells (Treg) has been progressing worldwide as a treatment for various autoimmune diseases, as regulatory T cells have the function of suppressing immune responses. In this way, regulatory T cells are expected to be used in the treatment and prevention of graft-versus-host disease (GvHD), autoimmune diseases, inflammatory diseases, and allergic diseases.

Regulatory T cells are broadly classified into two types: naturally occurring regulatory T cells (nTreg, or thymic Treg: tTreg) and inducible regulatory T cells (iTreg). nTregs are naturally generated in the thymus, while iTregs are known to be induced to differentiate from naive T cells in peripheral blood by antigen stimulation and cytokines such as IL-2 and TGF-β. These regulatory T cells are further subdivided according to the types of markers they express.

When regulatory T cells are removed from the body, various organ-specific autoimmune diseases spontaneously develop. However, transplanting regulatory T cells in such cases prevents the onset of autoimmune diseases. Therefore, regulatory T cells were thought to play an important role in maintaining immune self-tolerance in the periphery. It has since become clear that regulatory T cells can suppress not only autoimmunity, but also most immune responses, including inflammation caused by foreign antigens, transplant rejection, infection immunity, allergies, and tumor immunity. Furthermore, the transcription factor FOXP3 has now been identified as the master regulator of regulatory T cells.

FOXP3 is a master regulator of Tregs. It is known that FOXP3 is constitutively expressed in Tregs, but not in conventional T cells (also known as Tconv (conventional T cells), effector T cells, or inflammatory T cells) that do not have suppressive function. Research has been conducted on methods for efficiently expanding primary Tregs while maintaining FOXP3 expression, and compounds, cytokines, and combinations thereof that maintain expression and promote cell proliferation have been reported. Examples of such compounds and cytokines include IL-2 (Non-Patent Documents 1-7), IL-4 (Non-Patent Documents 2, 8, 9), and TNFR2 agonist antibodies (Non-Patent Documents 4, 5, 7, 10).

Further examples of such compounds, cytokines, and combinations thereof include IL-3 (Non-Patent Document 1) and TGF-β (Non-Patent Documents 1, 3, 6, 8, 11).

Although the effects of IL-2, IL-4, and TNFR2 agonists on primary Tregs have been reported as described above, there have been no reports suggesting that the combination of IL-2, IL-4, IL-33, and TNFR2 agonists (with the addition of IL-3 or TGF-β) is particularly preferable.

J Immunol (2011) 186:2262-2272 Immunology. 2009 Jul;127(3):338-344 PLoS One. , 11(2):e0148474. (2016) PLoS One. , 11(5), e0156311 (2016) Front. Immunol. 9:573 (2018) Clin. Exp. Immunol. , 197(1):52-63. (2019) Scientific Reports 3:3153 (2013) Immunology (2009) 128:e670-e678 Front. Immunol. 8:1508. doi:10.3389/fimmu. 2017.01508 Sci. Signal. 13, eaba9600 (2020) J Exp Med (2003) 198(12): 1875-1886

The present invention aims to provide a method for producing a cell population of expanded regulatory T cells, which can efficiently expand primary regulatory T cells, which are known to be difficult to expand, while maintaining their suppressive function.

As a result of extensive research conducted to achieve the above-mentioned objective, the inventors discovered that by culturing a cell population containing primary regulatory T cells in the presence of IL-2, IL-4, IL-33, and a TNFR2 agonist, regulatory T cells can be expanded with high efficiency while maintaining their suppressive function.

The present invention was completed based on these findings and through further investigation, and provides the following methods for producing cell populations in which regulatory T cells have expanded, cell populations containing regulatory T cells, pharmaceuticals, etc.

[1] A method for producing a cell population in which regulatory T cells have expanded, comprising:
(1) A method comprising the step of culturing a cell population containing primary regulatory T cells in the presence of interleukin (IL)-2, IL-4, IL-33 and a TNFR2 agonist.
[2] The step (1)
The method according to [1], further performed in the presence of a TGF-βR agonist.
[3] The step (1)
The method according to [1], further performed in the presence of IL-3.
[4] The step (1)
The method according to [1], further performed in the presence of IL-3 and a TGF-βR agonist.
[5] The method according to any one of [1] to [4], wherein the TNFR2 agonist is an anti-TNFR2 agonist antibody.
[6] The method according to [2], [4] or [5], wherein the TGF-βR agonist is TGF-β.
[7] The method according to any one of [1] to [6], wherein the culture in the step (1) is further carried out in the presence of at least one agonist selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist.
[7a] The method according to any one of [1] to [6], wherein in the culture of the step (1), the culture is further performed in the presence of at least one agonist selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist, and then in the absence of these agonists.
[8] The method according to any one of [1] to [7a], wherein the regulatory T cells are CD25 ⁺ /FOXP3 ⁺ cells.
[9] The method according to [8], wherein the CD25 ⁺ /FOXP3 ⁺ cells are CD4 ⁺ cells.
[9a] The method according to any one of [1] to [7a], wherein the regulatory T cells are CD25 ⁺ /CD127 ⁻ cells.
[9b] The method according to any one of [1] to [9a], wherein the regulatory T cells are Helios ⁺ cells.
[10] A method for expanding a cell population containing regulatory T cells, comprising:
(1A) A method comprising the step of culturing a cell population containing primary regulatory T cells in the presence of IL-2, IL-4, IL-33 and a TNFR2 agonist.
[10a] The step (1A)
The method according to [10], further performed in the presence of a TGF-βR agonist.
[10b] The step (1A)
The method according to [10], further performed in the presence of IL-3.
[10c] The step (1A)
The method according to [10], further performed in the presence of IL-3 and a TGF-βR agonist.
[10d] The method according to any one of [10] to [10c], wherein the TNFR2 agonist is an anti-TNFR2 agonist antibody.
[10e] The method according to [10a], [10c] or [10d], wherein the TGF-βR agonist is TGF-β.
[10f] The method according to any one of [10] to [10e], wherein the culture in the step (1A) is further carried out in the presence of at least one agonist selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist.
[10f1] The method according to any one of [10] to [10f], wherein in the culture of the step (1A), the culture is further performed in the presence of at least one agonist selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist, and then in the absence of these agonists.
[10g] The method according to any one of [10] to [10f1], wherein the regulatory T cells are CD25 ⁺ /FOXP3 ⁺ cells.
[10h] The method according to [10g], wherein the CD25 ⁺ /FOXP3 ⁺ cells are CD4 ⁺ cells.
[10i] The method according to any one of [10] to [10f1], wherein the regulatory T cells are CD25 ⁺ /CD127 ⁻ cells.
[10j] The method according to any one of [10] to [10i], wherein the regulatory T cells are Helios ⁺ cells.
[10k] (2) The method according to any one of [1] to [10j], comprising a step of introducing a foreign gene (particularly a gene for expressing a chimeric antigen receptor) into a cell population containing primary regulatory T cells or a cell population containing regulatory T cells obtained by the method according to any one of [1] to [10j].
[11] A cell population containing regulatory T cells obtained by the method according to any one of [1] to [10k].
[12] A pharmaceutical comprising a cell population containing regulatory T cells according to [11].
[13] The pharmaceutical agent according to [12], which is used for the prevention and/or treatment of abnormally enhanced immune response.
[14] A method for preventing and/or treating abnormally enhanced immune responses, comprising administering a cell population containing the regulatory T cells described in [11] to a subject in need thereof.
[15] A cell population comprising the regulatory T cells described in [11] for use in the prevention and/or treatment of abnormally enhanced immune responses.
[16] Use of a cell population containing regulatory T cells described in [11] in the manufacture of a pharmaceutical for preventing and/or treating abnormally enhanced immune responses.

The present invention makes it possible to proliferate primary regulatory T cells with high efficiency while maintaining their suppressive function.

This shows the results of isolating primary cultured Tregs from PBMCs using a flow cytometer. The numbers in the dot plots indicate the percentage of Tregs (CD4 ⁺ CD8α ⁻ CD25 ⁺ CD127 ⁻ ). 1 is a graph showing the change in cell number over time when primary cultured Tregs were expanded in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and anti-TNFR2 antibody. Optimized: Medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and anti-TNFR2 antibody. Control: Medium not containing IL-3, IL-4, IL-33, TGF-β1, or anti-TNFR2 antibody. This figure shows the results of examining the expression levels of proteins expressed in Tregs expanded in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and an anti-TNFR2 antibody. All dot plots in the figure represent the CD3 ⁺ CD4 ⁺ CD8 ⁻ population. The values on the graph indicate the proportion of Tregs (CD3 ⁺ CD4 ⁺ CD8 ⁻ CD25 ⁺ FOXP3 ⁺ ) in each cell population. The left figure shows the results of flow cytometry analysis of a cell population expanded in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and an anti-TNFR2 antibody after co-culture with allogeneic T cells derived from human PBMCs, and the right graph shows the inhibition rate (%) of target cell division. E:T indicates the ratio of Treg to human T cell numbers. FIG. 10 is a graph showing the change in cell number over time when primary cultured Tregs were expanded in a medium containing only IL-2, or in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and an anti-TNFR2 antibody from which any one of IL-3, IL-4, IL-33, and TGF-β1 had been removed. This figure shows the results of examining the expression levels of proteins expressed in Tregs expanded in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and an anti-TNFR2 antibody, but omitting any one of IL-3, IL-4, IL-33, and TGF-β1. All dot plots in the figure represent the CD3 ⁺ CD4 ⁺ CD8 ⁻ population. The values on the graph indicate the proportion of Tregs (CD3 ⁺ CD4 ⁺ CD8 ⁻ CD25 ⁺ FOXP3 ⁺ ) in each cell population. 1 is a graph showing the rate (%) of inhibition of target cell division after co-culture with human PBMC-derived allogeneic T cells using a cell population expanded in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and an anti-TNFR2 antibody but from which any one of IL-3, IL-4, IL-33, and TGF-β1 has been removed. E:T indicates the ratio of the number of Tregs to the number of human T cells. FIG. 1 is a graph showing the change in cell number over time when primary cultured Tregs were expanded in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and an anti-TNFR2 antibody, or in a medium containing the above medium composition minus IL-3, TGF-β1, and anti-TNFR2 antibody.

The following describes in detail the embodiments of the present invention.

"Comprise(s)" or "comprising" means the inclusion of, but is not limited to, the elements that follow the phrase. Thus, it implies the inclusion of the elements that follow the phrase, but does not imply the exclusion of any other elements. "Consist(s) of" means inclusive of and limited to all elements that follow the phrase. Thus, the phrase "consisting of" indicates that the listed elements are required or essential, and that other elements are substantially absent. "Consist(s) essentially of" means inclusive of any elements that follow the phrase, and is limited to other elements that do not affect the activity or function of the element identified in this disclosure. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or function of the listed elements.

As used herein, "culture" refers to maintaining and/or growing cells in an in vitro environment. "Culturing" refers to maintaining and/or growing cells outside a tissue or outside the body, for example, in a cell culture dish or flask.

As used herein, "culturing a cell population in the presence of a substance" refers to culturing a cell population in a medium containing the substance. Examples of such culturing include culturing in a medium containing the substance alone, or in which the substance coexists with other differentiation-inducing factors. When adding the substance to the medium, it can be added directly to the medium, or the substance can be dissolved in an appropriate solvent just before use and then added to the medium. The substance can also be immobilized on the surface of a substrate or carrier during culturing and then cultured.

As used herein, "positive (+)" means that the protein or gene is expressed in a detectable amount using techniques known in the art. In addition, in the case of proteins that are expressed intracellularly and not on the cell surface (e.g., transcription factors or their subunits), the target protein can be detected by expressing a reporter protein along with the protein and detecting the reporter protein. Gene detection can be performed using nucleic acid amplification and/or nucleic acid detection methods, such as RT-PCR, microarrays, biochips, and RNAseq.

As used herein, "negative (-)" means that the expression level of a protein or gene is below the lower limit of detection by any or all of the above-mentioned known techniques, or that the level of expression is low. The lower limit of detection for protein or gene expression may vary depending on the technique. The level of protein or gene expression (whether low or high) can be determined by comparing it with the results of control cells measured under the same conditions. For example, the level of CD25 expression in a certain cell population can be determined using flow cytometry, by comparing the CD25 expression level in that cell population with the CD25 expression level in PBMC-derived Tconv (control: known to be a low CD25 expressor). If expression equivalent to that of the control is observed, it is determined to be low expression, and if expression is higher than that of the control cells, it is determined to be high expression.

As used herein, the term "marker" refers to a protein or its gene that is specifically expressed on the cell surface, in the cytoplasm, in the nucleus, etc., of a specific cell type. The marker is preferably a "cell surface marker." A "cell surface marker" refers to a protein expressed on the cell surface that can be labeled (stained) with a fluorescent substance, making it easy to detect, concentrate, isolate, etc., cells expressing the cell surface marker. The cell surface marker refers to a gene that is specifically expressed (positive marker) or not expressed (negative marker) in a specific cell type; specifically, a substance that is produced (positive marker) or not produced (negative marker) as mRNA resulting from transcription of the gene in the genome, or as a protein resulting from translation of that mRNA.

Such cell surface markers can be detected using immunological assays using antibodies specific to the cell surface markers, such as ELISA, immunostaining, and flow cytometry.

As used herein, "expression" is defined as the transcription and/or translation of a specific nucleotide sequence driven by a promoter within a cell.

As used herein, the term "regulatory T cells" refers to T cells that, upon stimulation via a T cell receptor, have the ability to inhibit the activation of effector T cells and are responsible for suppressing immune responses (immune tolerance). Regulatory T cells are usually CD25 ⁺ /FOXP3 ⁺ cells or CD25 ⁺ /CD127 ^- cells, among which CD4 ⁺ or CD8 ⁺ cells are present. In the present invention, regulatory T cells may be CD4 ⁺ cells (i.e., CD4 ⁺ /CD25 ⁺ /FOXP3 ⁺ or CD4 ⁺ /CD25 ⁺ /CD127 ^- ) or CD8 ⁺ cells (i.e., CD8 ⁺ /CD25 ⁺ /FOXP3 ⁺ or CD8 ⁺ /CD25 ⁺ /CD127 ^- ), with CD4 ⁺ cells being more preferred. Furthermore, the transcription factor FOXP3 is known as a master regulator of CD4 ⁺ /CD25 ⁺ regulatory T cells. Regulatory T cells may also be positive for Helios, CTLA4 (Cytotoxic T Lymphocyte (associated) Antigen 4) (CD152), CD39, and CD73, which are known to be indicators of the suppressive function of regulatory T cells. Helios is a member of the Ikaros transcription factor family and has been shown to bind to the FOXP3 promoter and increase FOXP3 expression. CTLA4 is an immune checkpoint protein, and when CTLA4 expressed on the surface of T cells binds to CD80 or CD86 on the surface of antigen-presenting cells, T cell activation is suppressed. It is also known that CD39 and CD73 hydrolyze ATP and AMP, respectively, to produce adenosine as the final product, and that the activation of T cells is suppressed when adenosine acts on T cells. In this specification, the "/" used in expressions such as "CD25 ⁺ /FOXP3 ⁺ " means "and."

As used herein, "cell population" refers to two or more cells of the same or different types. "Cell population" also refers to a mass of cells of the same or different types.

As used herein, "regulatory T cells expanded" and "expansion of regulatory T cells" refer to an increase in the number (absolute number) of regulatory T cells in a cell population compared to before culture or a control such as cells obtained without carrying out the present invention, and mean, for example, an increase of at least 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 300-fold, 500-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10000-fold, 15000-fold, 20000-fold, 50000-fold, or 100000-fold compared to before culture or the control. In certain embodiments of the present invention, the number (absolute number) of regulatory T cells in a cell population produced by the present invention is increased by at least 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 300-fold, 500-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10000-fold, 15000-fold, 20000-fold, 50000-fold, or 100000-fold compared to the number of cells before culture or in a control.

From the perspective of therapeutic application, it is preferable that the various cells used in the present invention are GMP (Good Manufacturing Practice) standard cells.

"Nucleic acid" refers to any molecule formed by polymerizing nucleotides and molecules with equivalent functions to the nucleotides. Examples include RNA, which is a polymer of ribonucleotides; DNA, which is a polymer of deoxyribonucleotides; mixed polymers of ribonucleotides and deoxyribonucleotides; and nucleotide polymers containing nucleotide analogs. Nucleic acids may also be nucleotide polymers containing nucleic acid derivatives. Nucleic acids may be single-stranded or double-stranded. Double-stranded nucleic acids also include double-stranded nucleic acids in which one strand hybridizes to the other strand under stringent conditions.

Nucleotide analogs may be any molecule in which ribonucleotides, deoxyribonucleotides, RNA, or DNA have been modified to improve nuclease resistance or stabilization compared to RNA or DNA, increase affinity with complementary nucleic acids, increase cell permeability, or enable visualization. Nucleotide analogs may be naturally occurring or unnatural molecules, such as sugar-modified nucleotide analogs (e.g., nucleotide analogs substituted with 2'-O-methylribose, nucleotide analogs substituted with 2'-O-propylribose, nucleotide analogs substituted with 2'-methoxyethoxyribose, nucleotide analogs substituted with 2'-O-methoxyethylribose, nucleotide analogs substituted with 2'-O-[2-(guanidinium)ethyl]ribose, nucleotide analogs substituted with 2'-fluororibose, and bridged artificial nucleotides. Examples include nucleic acids (BNA: Bridged Nucleic Acid), locked artificial nucleic acids (LNA: Locked Nucleic Acid), ethylene-bridged artificial nucleic acids (ENA: Ethylene-bridged nucleic acid), peptide nucleic acids (PNA), oxypeptide nucleic acids (OPNA), peptide ribonucleic acids (PRNA), nucleotide analogs modified with phosphodiester bonds (e.g., nucleotide analogs substituted with phosphorothioate bonds, nucleotide analogs substituted with N3'-P5' phosphoamidate bonds), etc.

A nucleic acid derivative can be any molecule in which another chemical substance has been added to the nucleic acid in order to improve nuclease resistance, stabilization, affinity with complementary nucleic acid strands, cell permeability, or visualization, compared to nucleic acids. Specific examples include 5'-polyamine-added derivatives, cholesterol-added derivatives, steroid-added derivatives, bile acid-added derivatives, vitamin-added derivatives, Cy5-added derivatives, Cy3-added derivatives, 6-FAM-added derivatives, and biotin-added derivatives.

The method of the present invention for producing a cell population in which regulatory T cells have expanded (sometimes referred to herein simply as the "production method of the present invention") is characterized by comprising the following steps:
(1) Culturing a cell population containing primary regulatory T cells in the presence of interleukin (IL)-2, IL-4, IL-33, and a TNFR2 agonist.

Step (1) can also be carried out in the presence of a TGF-βR agonist and/or IL-3 in addition to IL-2, IL-4, IL-33, and a TNFR2 agonist. That is, step (1) can be carried out in the presence of IL-2, IL-3, IL-4, IL-33, and a TNFR2 agonist; in the presence of IL-2, IL-4, IL-33, a TGF-βR agonist, and a TNFR2 agonist; or in the presence of IL-2, IL-3, IL-4, IL-33, a TGF-βR agonist, and a TNFR2 agonist. From the perspective of regulatory T cell proliferation, step (1) is preferably carried out in the absence of an mTOR inhibitor (e.g., rapamycin).

IL-4, IL-3, IL-2, and IL-33 are preferably derived from mammals, and particularly preferably from humans. IL-2, IL-3, IL-4, and IL-33 may be natural products isolated and purified from mammals (particularly humans), or may be artificially produced using genetic engineering techniques (which may include amino acid substitutions, deletions, insertions, and/or additions).

A TGF-βR (Transforming Growth Factor-β Receptor) agonist is defined as a substance that can bind to TGF-βR and activate TGF-βR signal transduction. Examples of TGF-βR agonists include factors belonging to the TGF-β superfamily, which includes the TGF-β family, the activin family, and the BMP (bone morphogenetic protein) family. Factors belonging to the TGF-β superfamily include, for example, TGF-β (TGF-β1, TGF-β2, TGF-β3), activin A, activin B, GDF-8, and GDF-11. A preferred TGF-βR agonist is TGF-β, and more preferably TGF-β1. TGF-βR agonists can be used singly or in combination of two or more.

TNFR2 (Tumor Necrosis Factor Receptor-2) agonists are defined as substances that can bind to TNFR2 and activate TNFR2 signaling, and include, for example, antibodies (e.g., anti-TNFR2 antibodies), peptides, small molecules, and proteins. Examples of anti-TNFR2 agonist antibodies include monoclonal antibodies that bind to TNFR2, such as clone MR2-1 (Hycult Biotech) and clone MAB2261 (R&D Systems). TNFR2 agonists may also be TNF-α muteins that bind only to TNFR2 as agonists. TNFR2 agonists can be used singly or in combination of two or more.

The TNFR2 agonist is preferably an anti-TNFR2 agonist antibody. The antibody may be a functional fragment thereof, such as Fd, Fv, Fab, F(ab'), F(ab) ₂ , F(ab') ₂ , single-chain Fv (scFv), diabody, triabody, tetrabody, and minibody. Antibodies include those derived from animals such as mouse, rat, cow, rabbit, goat, sheep, and guinea pig. The antibody isotype is not particularly limited, and examples of isotypes include IgG (IgG1, IgG2, IgG3, IgG4), IgA, IgD, IgE, and IgM. Furthermore, the antibody may be either a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody, and may also be a humanized antibody, a chimeric antibody, a multispecific antibody (e.g., a bispecific antibody), etc. Antibodies can be produced by known methods, for example, by constructing an expression vector containing a nucleic acid encoding the antibody, culturing a transformant into which the nucleic acid has been introduced, or culturing a hybridoma that produces the antibody.

IL-2, IL-3, IL-4, IL-33, TGF-βR agonists, and TNFR2 agonists can be used in their free state or in the form of a salt. Examples of salts include salts with inorganic bases such as sodium salt, magnesium salt, potassium salt, calcium salt, and aluminum salt; salts with organic bases such as methylamine salt, ethylamine salt, and ethanolamine salt; salts with basic amino acids such as lysine, ornithine, and arginine; and ammonium salts. The salts may also be acid addition salts, and specific examples of such salts include acid addition salts with mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, malic acid, tartaric acid, fumaric acid, succinic acid, lactic acid, maleic acid, citric acid, methanesulfonic acid, and ethanesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid. IL-2, IL-3, IL-4, IL-33, TGF-βR agonists, and TNFR2 agonists also include hydrates, solvates, and crystalline polymorphs.

The concentration of IL-2 in the culture medium is not particularly limited, and is, for example, 0.1 to 1000 ng/mL, preferably 10 to 500 ng/mL, and more preferably 50 to 200 ng/mL.

The concentration of IL-4 in the culture medium is not particularly limited, and is, for example, 0.01 to 100 ng/mL, preferably 10 to 100 ng/mL.

The concentration of IL-33 in the culture medium is not particularly limited, and is, for example, 0.1 to 100 ng/mL, preferably 10 to 100 ng/mL.

The concentration of the TNFR2 agonist in the medium is not particularly limited and can be adjusted appropriately depending on the type of TNFR2 agonist used. The concentration of the TNFR2 agonist is, for example, 0.0001 to 100 μg/mL, preferably 0.01 to 10 μg/mL.

The concentration of the TGF-βR agonist in the culture medium is not particularly limited and can be adjusted appropriately depending on the type of TGF-βR agonist used. The concentration of the TGF-βR agonist is, for example, 0.1 to 100 ng/mL, preferably 1 to 50 ng/mL.

The concentration of IL-3 in the culture medium is not particularly limited, and is, for example, 0.01 to 100 ng/mL, preferably 10 to 100 ng/mL.

The medium used in the culture of step (1) is a basal medium that is used for culturing animal cells and contains the aforementioned IL-2, IL-4, IL-33, and TNFR2 agonist (as well as IL-3, TGF-βR agonist, etc.). The basal medium is not particularly limited as long as it can be used for culturing animal cells, and examples include AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, 199 Medium, Eagle's MEM, αMEM, DMEM, Ham, RPMI-1640, and Fischer's Medium. Any one of these media may be used alone, or two or more may be used in combination.

The medium may contain serum or may be serum-free. The medium may also contain serum substitutes (e.g., albumin, transferrin, Knockout Serum Replacement (KSR), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, ITS supplement, B27™ supplement, etc.). One or more types of serum substitutes may be used.

Furthermore, the medium may contain one or more substances such as lipids, amino acids (non-essential amino acids, etc.), L-glutamine, vitamins, growth factors, cytokines, anti-CD3 antibodies, anti-CD30 antibodies, anti-CD28 antibodies, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. It is preferable to use a chemically defined medium that does not contain unknown components such as serum, as this reduces differences between medium lots and allows for the preparation of cells of consistent quality.

The pH of the medium is usually 7.0 to 7.8, preferably 7.2 to 7.6. Prior to use, the medium is preferably sterilized by filtration, ultraviolet irradiation, heat sterilization, radiation, or other methods to prevent contamination.

Culturing can be carried out in the presence or absence of feeder cells. The production method of the present invention is preferably carried out in the absence of feeder cells, as it allows for the stable production of regulatory T cells with uniform properties without contamination by unknown components.

The culture conditions for the production method of the present invention are not particularly limited. The culture temperature is, for example, about 37 to 42°C, preferably about 37 to 39°C, the _CO2 concentration is, for example, 2 to 10%, preferably 2 to 5%, and the oxygen concentration is, for example, 1 to 20%, preferably 5 to 20%.

The culture period is also not particularly limited and can be appropriately determined by those skilled in the art while monitoring the number of regulatory T cells, etc., and is, for example, 7 days or more, preferably 14 days or more, more preferably 21 days or more, and even more preferably 28 days or more. The upper limit of the culture period is not particularly limited and, for example, 42 days or less, preferably 35 days or less, and more preferably 28 days or less. In the culture of the present invention, passages may be performed as many times as necessary to obtain the desired amount of regulatory T cells, and medium addition and replacement may be performed. The culture of the present invention can be performed using a known _CO2 incubator. The culture vessel is not particularly limited and can be appropriately selected from plates, dishes, petri dishes, flasks, bags, bottles, tanks (culture vessels), bioreactors, etc. A vessel to which an anti-CD3 antibody is bound can be used as the culture vessel.

In step (1), culture (cell stimulation) may be performed in the presence of IL-2, IL-4, IL-33, and a TNFR2 agonist (IL-3 and a TGF-βR agonist may also be added), and in the presence of at least one selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist. More preferably, culture (cell stimulation) may be initially initiated in the presence of IL-2, IL-4, IL-33, and a TNFR2 agonist (IL-3 and a TGF-βR agonist may also be added), and in the presence of at least one selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist, and then culture may be performed in the absence of these antibodies. Step (1) may be repeated (by subculture). The culture may be performed by first initiating culture (stimulating the cells) in the presence of at least one selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist, followed by repeated culture in the absence of these antibodies. The culture period for this repetition is, for example, 7 to 28 days, preferably 14 to 21 days. Specifically, the cells may be cultured (stimulated) in the presence of at least one selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist for the first 3 days or so, followed by culture in a medium that does not contain these agonists for 4 to 11 days, and this culture unit may be repeated. The number of culture repetitions is not particularly limited and can be determined appropriately by a person skilled in the art while monitoring the number of immune cells of the present invention, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more. The upper limit of the number of culture repetitions is, for example, 20 times.

The CD30 agonist is not particularly limited as long as it is a molecule that can transmit a signal from CD30 into the cell by specifically binding to CD30. Examples of CD30 agonists include anti-CD30 agonist antibodies or functional fragments thereof, and CD30 ligands or functional fragments thereof. The concentration of the CD30 agonist in the culture medium is not particularly limited and is adjusted appropriately depending on the type of CD30 agonist used. The concentration of the CD30 agonist is, for example, 10 to 1000 ng/mL, preferably 100 to 500 ng/mL.

The CD3 agonist is not particularly limited, as long as it is a molecule that can transmit a signal from CD3 into the cell by specifically binding to CD3. Examples of CD3 agonists include anti-CD3 agonist antibodies or functional fragments thereof, and CD3 ligands or functional fragments thereof. The concentration of the CD3 agonist in the culture medium is not particularly limited and is adjusted appropriately depending on the type of CD3 agonist used. The concentration of the CD3 agonist is, for example, 10 to 10,000 ng/mL, preferably 100 to 10,000 ng/mL, and more preferably 1,000 to 5,000 ng/mL.

The CD28 agonist is not particularly limited, as long as it is a molecule that can transmit a signal from CD28 into the cell by specifically binding to CD28. Examples of CD28 agonists include anti-CD28 agonist antibodies or functional fragments thereof, and CD28 ligands or functional fragments thereof. The concentration of the CD28 agonist in the culture medium is not particularly limited and is adjusted appropriately depending on the type of CD28 agonist used. The concentration of the CD28 agonist is, for example, 10 to 10,000 ng/mL, preferably 100 to 10,000 ng/mL, and more preferably 500 to 5,000 ng/mL.

The cell population containing regulatory T cells used in the production method of the present invention is primary cultured cells isolated from biological tissues such as bone marrow, umbilical cord blood, and blood. Primary cultured cells refer to cells that have been isolated from organs, cells, and tissues separated from a living body and seeded until the first passage. Primary cultured cells can be obtained from organs and tissues by known methods such as enzyme treatment, physical dispersion, and the explant method. The proportion (cell number) of regulatory T cells contained in the cell population containing primary regulatory T cells is, for example, 80% or more, preferably 90% or more, more preferably 95% or more, with an upper limit of, for example, 100% or less. Regulatory T cells can be isolated from cells separated from biological tissues by known methods such as flow cytometry or magnetic cell separation.

The cell population containing primary regulatory T cells used in the production method of the present invention may be derived from humans or from mammals other than humans (non-human mammals), preferably humans. Examples of non-human mammals include mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, pigs, cows, horses, sheep, and monkeys.

The production method of the present invention may further include a step of separating regulatory T cells in order to enrich the obtained regulatory T cells. Separation of regulatory T cells can be carried out by known methods such as flow cytometry or magnetic cell separation.

The manufacturing method of the present invention makes it possible to produce a cell population containing regulatory T cells in which regulatory T cells have proliferated. In other words, the manufacturing method of the present invention makes it possible to expand and culture regulatory T cells.

Furthermore, another embodiment of the present invention, a method for expanding a cell population containing regulatory T cells (sometimes referred to herein simply as the "expansion method of the present invention"), is characterized by comprising the following steps:
(1A) Culturing a cell population containing primary regulatory T cells in the presence of IL-2, IL-4, IL-33 and a TNFR2 agonist.

Step (1A) can be carried out in the same manner as step (1). The explanation regarding the production method of the present invention applies to the method for expanding the above-mentioned cell population containing regulatory T cells.

The regulatory T cells obtained by the production method and proliferation method of the present invention may be regulatory T cells into which an exogenous gene has been introduced.

An "exogenous gene" is a gene introduced from outside to cause regulatory T cells to express a desired protein, and can be selected appropriately depending on the intended use of the regulatory T cells.

The foreign gene can be, for example, a gene for expressing a chimeric antigen receptor (CAR), which can further include genes for expressing cytokines and/or chemokines. Similar to common or known CARs, the CAR expressed by regulatory T cells is essentially composed of peptides from each of the following sites, linked, as necessary, via a spacer: (i) an antigen recognition site (e.g., a single-chain antibody) that recognizes a cell surface antigen on a cancer cell; (ii) a cell membrane-spanning domain; and (iii) a signal transduction domain that induces T cell activation. The foreign gene can also be, for example, a gene for expressing an exogenous T cell receptor (TCR). An exogenous TCR means that the nucleic acid encoding the exogenous TCR is exogenous to the T cell into which it is introduced, and the amino acid sequence of the exogenous TCR may be the same as or different from the endogenous TCR of the T cell. One or more types of foreign genes can be introduced (e.g., a CAR and an exogenous TCR).

The means for introducing a foreign gene into cells is not particularly limited, and various known or common means can be used. Typically, the foreign gene is introduced into cells using an expression vector and expressed. The expression vector may be linear or circular, and may be a non-viral vector such as a plasmid, a viral vector, or a transposon-based vector. The cells into which the foreign gene is introduced are not particularly limited, and may be at any stage. Examples include primary regulatory T cells before carrying out step (1) or (1A), and regulatory T cells obtained by the production method and expansion method of the present invention.

The technique for introducing an expression vector into cells can be an appropriate one depending on the embodiment. For example, the expression vector can be introduced into cells by known methods such as viral infection, calcium phosphate precipitation, lipofection, microinjection, and electroporation. The expression vector can be prepared in a form suitable for use in each technique by known means or, as appropriate, using a commercially available kit (following the instructions).

The expression vector can be introduced into cells by viral infection. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors. When using these viral vectors, a corresponding commercially available kit can be used to transfect host cells with the vector containing the foreign gene and the packaging vector (plasmid) of each virus to produce a recombinant virus, and then the resulting recombinant virus can be used to infect cells.

In addition to the foreign gene, the expression vector may contain sequences such as a nuclear localization signal (NLS) or a multiple cloning site (MCS) as necessary. The expression vector may further contain a reporter gene (e.g., a gene encoding a fluorescent protein of each color), a drug selection gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene), a suicide gene (e.g., a diphtheria A toxin, a herpes simplex thymidine kinase (HSV-TK), a carboxypeptidase G2 (CPG2), a carboxylesterase (CA), a cytosine deaminase (CD), a cytochrome P450 (cyt-450), It may also contain nucleic acids (base sequences) encoding "functional genes" such as genes encoding deoxycytidine kinase (dCK), nitroreductase (NR), purine nucleoside phosphorylase (PNP), thymidine phosphorylase (TP), varicella-zoster virus thymidine kinase (VZV-TK), xanthine-guanine phosphoribosyltransferase (XGPRT), inducible caspase 9, etc.

A cell population containing regulatory T cells produced by the production method and expansion method of the present invention is useful for treating animals (particularly humans) with abnormally enhanced immune responses, including, but not limited to, X-linked immunodysregulation/polyendocrinopathy/enteropathy (IPEX) syndrome, graft-versus-host disease (GVHD), organ transplant rejection, autoimmune diseases, inflammatory diseases, and allergic diseases (hay fever, asthma, atopic dermatitis, eczema, food allergies, food hypersensitivity, urticaria, allergic rhinitis, allergic conjunctivitis, and drug allergies). Regulatory T cells produced by the production method and expansion method of the present invention may be used for autologous or allogeneic transplantation. They may also be used in combination with other drugs.

When carrying out such cell therapy, in order to avoid rejection reactions, it is preferable that the subject from whom the cells to be used in producing regulatory T cells are isolated has an HLA type that matches that of the subject to which the regulatory T cells will be administered, and it is even more preferable that the subject is the same as the subject to which the regulatory T cells will be administered.

According to the present invention, it is possible to produce a pharmaceutical comprising a cell population that includes regulatory T cells (hereinafter, sometimes referred to as the pharmaceutical of the present invention). The pharmaceutical of the present invention is preferably produced as a parenteral formulation by mixing an effective amount of regulatory T cells with a pharmaceutically acceptable carrier according to known means (for example, the method described in the Japanese Pharmacopoeia). The pharmaceutical of the present invention is preferably produced as a parenteral formulation such as an injection, suspension, or infusion. Parenteral administration methods include intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration. Pharmaceutically acceptable carriers include, for example, solvents, bases, diluents, excipients, soothing agents, buffers, preservatives, stabilizers, suspending agents, isotonicity agents, surfactants, and solubilizing agents.

The dosage of the medicament of the present invention can be determined appropriately depending on various conditions, such as the patient's body weight, age, sex, and symptoms. Typically, the number of cells administered per administration to a subject weighing 60 kg is typically 1×10 ⁶ to 1×10 ¹⁰ , preferably 1×10 ⁷ to 1×10 ⁹ , and more preferably 5×10 ⁷ to 5×10 ^8. The medicament may be administered once or multiple times. The medicament of the present invention can be in a known form suitable for parenteral administration, such as an injection or infusion. Furthermore, the medicament of the present invention may contain physiological saline, phosphate-buffered saline (PBS), a culture medium, or the like, to stably maintain the cells. Examples of culture media include, but are not limited to, RPMI, AIM-V, and X-VIVO10. Furthermore, the medicament may contain a pharmaceutically acceptable carrier (e.g., human serum albumin), a preservative, or the like, for stabilization purposes. The medicament of the present invention is intended for use in mammals, including humans.

As used in this specification and claims, singular terms shall include plurals and plural terms shall include the singular, unless the context otherwise requires. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept, unless otherwise specified.

The following examples are provided to further explain the present invention. However, the present invention is not limited to these examples.

(1) Isolation of primary cultured Tregs CD4 ⁺ T cells were isolated from human peripheral blood mononuclear cells (PBMCs (HemaCare)) according to the recommended protocol of the CD4 ⁺ T cell isolation kit Human (Myltenyi Biotec). Then, they were stained with the following antibody set and analyzed by flow cytometry, and Tregs (CD4 ⁺ CD8α ^- CD25 ⁺ CD127 ^- ) were purified. The results are shown in Figure 1.
PE/Cy7 CD4, PerCP/Cy5.5 CD8α, PE CD127, APC CD25.

Purified Tregs were obtained with a purity of 99.0%.

(2) Expansion of Primary Cultured Tregs The primary cultured Tregs obtained in (1) were seeded at 1 x ¹⁰ cells/well onto a 96-well cell culture plate conjugated with anti-CD3 antibody (3 μg/mL, eBioscience) and cultured for 3 days at 37°C and 5.0% CO in _{a CO} incubator. After further culture for 3 days, the cells were seeded onto a 48-well cell culture plate and continued to be cultured. After further culture for 3 days, the cells were seeded onto a 24-well G-Rex cell culture plate and continued to be cultured. The medium contained, at final concentrations, FBS (15%, Corning), L-glutamine-penicillin-streptomycin solution (1/100, Invitrogen, Sigma-Aldrich), insulin-transferrin-selenium supplement (1/100, Invitrogen), ascorbic acid 2-phosphate (50 μg/mL, Sigma-Aldrich), IL-2 (100 ng/mL, PeproTech), and anti-TNFR2 antibody (3 μg/mL, Hycult The α-MEM (Invitrogen) medium used contained IL-1 (60 ng/mL, BioLegend), IL-3 (60 ng/mL, BioLegend), IL-4 (30 ng/mL, BioLegend), IL-33 (30 ng/mL, BioLegend), and TGF-β1 (5 ng/mL, BioLegend). For the first three days, anti-CD28 antibody (1.5 μg/mL, BioLegend) and anti-CD30 antibody (300 ng/mL, R&D Systems) were further added to the above composition. A control group used medium containing the above medium composition but omitting anti-TNFR2 antibody, IL-3, IL-4, IL-33, and TGF-β1. The cells were cultured for 15 days, with the medium being changed on days 3, 6, 8, 11, and 13 after the start of culture, and the number of cells was counted and a line graph was created. The results are shown in Figure 2.

The cell population obtained through expansion culture showed a proliferation rate of approximately 5,700 times on the 14th day of culture. Furthermore, proliferation was enhanced compared to the control group.

(3) Examination of Treg protein expression levels during expansion culture. Each cell population, which was expanded in (2) in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and anti-TNFR2 antibody, was used on day 15 after the start of culture. The cells were stained with the following antibody set and analyzed by flow cytometry. The results are shown in Figure 3: Zombie NIR, BV510 CD3, BV421 CD4, PE/Cy7 CD8β, APC CD25, FITC FOXP3, PE Helios.

The cell population obtained using the above method showed a FOXP3 positivity rate of 77.6%. The Helios positivity rate was also 59.5%.

(4) Evaluation of the suppressive function of Tregs. PBMCs derived from a donor other than Tregs and untreated or treated with anti-CD3 antibody were stained with CellTrace Violet (Thermo Fisher Scientific) and used as target cells. These cells were co-cultured with Tregs expanded in a medium containing IL-2, IL-3, IL-4, IL-33, TGF-β1, and anti-TNFR2 antibody in (2). The cells were cultured for 4 days in α-MEM (Invitrogen) containing final concentrations of FBS (15%, Corning), L-glutamine-penicillin-streptomycin solution (1/100, Invitrogen, Sigma-Aldrich), insulin-transferrin-selenium supplement (1/100, Invitrogen), and ascorbic acid 2-phosphate (50 μg/mL, Sigma-Aldrich). Each cell population was stained with the following antibody set. The results are shown in Figure 4.
Zombie NIR, PE/Cy7 CD3, FITC HLA-A24.

A strong suppression of target cell (CD3 ⁺ ) cell division was observed in a Treg cell number-dependent manner.

(5) Expansion of Primary Cultured Tregs The primary cultured Tregs obtained in (1) were seeded at 1 x ¹⁰ cells/well onto a 96-well cell culture plate conjugated with anti-CD3 antibody (3 μg/mL, eBioscience) and cultured for 3 days at 37°C and 5.0% CO in _{a CO} incubator. After further culture for 3 days, the cells were seeded onto a 48-well cell culture plate and continued to be cultured. After further culture for 3 days, the cells were seeded onto a 24-well G-Rex cell culture plate and continued to be cultured. The medium contained, at final concentrations, FBS (15%, Corning), L-glutamine-penicillin-streptomycin solution (1/100, Invitrogen, Sigma-Aldrich), insulin-transferrin-selenium supplement (1/100, Invitrogen), ascorbic acid 2-phosphate (50 μg/mL, Sigma-Aldrich), IL-2 (100 ng/mL, PeproTech), and anti-TNFR2 antibody (3 μg/mL, Hycult). The culture medium used was α-MEM (Invitrogen) containing IL-3 (60 ng/mL, BioLegend), IL-4 (30 ng/mL, BioLegend), IL-33 (30 ng/mL, BioLegend), and TGF-β1 (5 ng/mL, BioLegend). For the first 3 days, anti-CD28 antibody (1.5 μg/mL, BioLegend) and anti-CD30 antibody (300 ng/mL, R&D Systems) were further added to the above composition. However, in practice, a medium containing IL-2 but not IL-3, IL-4, IL-33, TGF-β1, or anti-TNFR2 antibody (medium containing only IL-2) was used, or a medium obtained by removing any one of IL-3, IL-4, IL-33, and TGF-β1 from the medium composition. The culture was continued for 14 days, with medium changes on days 3, 6, 8, 11, and 13 after the start of culture, and the number of cells was counted and a line graph was created. The results are shown in FIG. 5.

The cell population obtained through expansion culture showed reduced proliferation ability on the 14th day of culture when IL-4 and IL-2 were removed from the above medium conditions.

(6) Examination of Treg protein expression levels during expansion culture. Each cell population expanded in (5) on day 14 after the start of culture (excluding the cell collection expanded in medium containing only IL-2, which could not be maintained until day 14 after the start of culture) was stained with the following antibody set and analyzed by flow cytometry. The results are shown in Figure 6: Zombie NIR, BV510 CD3, BV421 CD4, PE/Cy7 CD8β, APC CD25, FITC FOXP3.

When the CD25 + FOXP3 + population was expanded in a medium containing the cytokines IL-3, IL-4, IL-33, and TGF-β1 from the medium composition of (5), the percentages of the CD25 ⁺ FOXP3 ⁺ population in each cell population were 62.8%, 50.5%, 65.8%, and 30.7%, respectively.

(7) Evaluation of the suppressive function of Tregs. PBMCs derived from a donor other than Tregs and untreated or treated with anti-CD3 antibodies were stained with CellTrace Violet (Thermo Fisher Scientific) and used as target cells. These cells were co-cultured with Tregs expanded in a medium containing no cytokines (IL-3, IL-4, IL-33, or TGF-β1) as described in (5). The cells were cultured for 4 days in α-MEM (Invitrogen) containing final concentrations of FBS (15%, Corning), L-glutamine-penicillin-streptomycin solution (1/100, Invitrogen, Sigma-Aldrich), insulin-transferrin-selenium supplement (1/100, Invitrogen), and ascorbic acid 2-phosphate (50 μg/mL, Sigma-Aldrich). Each cell population was stained with the following antibody set. The results are shown in Figure 7.
Zombie NIR, PE/Cy7 CD3, FITC HLA-A24.

A strong suppression of target cell (CD3 ⁺ ) cell division was observed in a Treg cell number-dependent manner.

(8) Expansion of Primary Cultured Tregs The primary cultured Tregs obtained in (1) were seeded at 1 x ¹⁰ cells/well onto a 96-well cell culture plate conjugated with anti-CD3 antibody (3 μg/mL, eBioscience) and cultured for 3 days at 37°C and 5.0% CO in _{a CO} incubator. After further culture for 3 days, the cells were seeded onto a 48-well cell culture plate and continued to be cultured. After further culture for 3 days, the cells were seeded onto a 24-well G-Rex cell culture plate and continued to be cultured. The medium contained, at final concentrations, FBS (15%, Corning), L-glutamine-penicillin-streptomycin solution (1/100, Invitrogen, Sigma-Aldrich), insulin-transferrin-selenium supplement (1/100, Invitrogen), ascorbic acid 2-phosphate (50 μg/mL, Sigma-Aldrich), IL-2 (100 ng/mL, PeproTech), and anti-TNFR2 antibody (3 μg/mL, Hycult The culture medium used was α-MEM (Invitrogen) containing IL-3 (60 ng/mL, BioLegend), IL-4 (30 ng/mL, BioLegend), IL-33 (30 ng/mL, BioLegend), and TGF-β1 (5 ng/mL, BioLegend). For the first three days, anti-CD28 antibody (1.5 μg/mL, BioLegend) and anti-CD30 antibody (300 ng/mL, R&D Systems) were further added to the above composition. A medium containing the above medium composition but omitting IL-3, TGF-β1, and anti-TNFR2 antibody was also used. The culture was continued for 22 days, with the medium being changed on days 3, 6, 8, 11, 13, 15, 18, and 20 after the start of culture, and the number of cells was counted and a line graph was created. The results are shown in Figure 8.

The above medium conditions resulted in stronger proliferation than medium conditions in which IL-3, TGF-β1, and anti-TNFR2 antibody were omitted.

This application is based on Japanese Patent Application No. 2024-048579, filed on March 25, 2024, the contents of which are incorporated in their entirety herein.

Claims (16)

A method for producing a cell population in which regulatory T cells are expanded, comprising:
(1) A method comprising the step of culturing a cell population containing primary regulatory T cells in the presence of interleukin (IL)-2, IL-4, IL-33 and a TNFR2 agonist. The step (1)
The method according to claim 1, further carried out in the presence of a TGF-βR agonist. The step (1)
The method according to claim 1, further carried out in the presence of IL-3. The step (1)
The method according to claim 1, further carried out in the presence of IL-3 and a TGF-βR agonist. The method of claim 1, wherein the TNFR2 agonist is an anti-TNFR2 agonist antibody. The method of claim 2 or 4, wherein the TGF-βR agonist is TGF-β. The method of claim 1, wherein the culture in step (1) is further performed in the presence of at least one agonist selected from the group consisting of a CD30 agonist, a CD3 agonist, and a CD28 agonist. The method of claim 1 , wherein the regulatory T cells are CD25 ⁺ /FOXP3 ⁺ cells. The method of claim 8, wherein the CD25 ⁺ /FOXP3 ⁺ cells are CD4 ⁺ . A method for expanding a cell population containing regulatory T cells, comprising:
(1A) A method comprising the step of culturing a cell population containing primary regulatory T cells in the presence of IL-2, IL-4, IL-33 and a TNFR2 agonist. A cell population containing regulatory T cells obtained by the method of claim 1 or 10. A pharmaceutical comprising a cell population containing the regulatory T cells described in claim 11. The pharmaceutical agent described in claim 12 for use in the prevention and/or treatment of abnormally enhanced immune responses. A method for preventing and/or treating abnormally enhanced immune responses, comprising administering a cell population containing the regulatory T cells described in claim 11 to a subject in need thereof. A cell population comprising the regulatory T cells of claim 11 for use in the prevention and/or treatment of abnormally enhanced immune responses. Use of a cell population containing regulatory T cells described in claim 11 in the manufacture of a medicament for preventing and/or treating abnormally enhanced immune responses.